The present invention relates the proteins having biological activity in plant and animal systems, to polynucleotides encoding for the expression of such proteins, to oligonucleotides for use in identifying and synthesizing these proteins and polynucleotides, to vectors and cells containing the polynucleotides in recombinant form and to plants and animals comprising these, and to the use of the proteins and polynucleotides and fragments thereof in the control of plant growth and plant vulnerability to viruses.
Cell cycle progression is regulated by positive and negative effectors. Among the latter, the product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. In mammalian cells, Rb regulates G1/S transit by inhibiting the function of the E2F family of transcription factors, known to interact with sequences in the promoter region of genes required for cellular DNA replication (see eg Weinberg, R. A. Cell 81,323 (1995); Nevins, J. R. Science 258,424 (1992)). DNA tumor viruses that infect animal cells express oncoproteins that interact with the Rb protein via a LXCXE motif, disrupting Rb-E2F complexes and driving cells into S-phase (Weinberg ibid; Ludlow, J. W. FASEB J. 7, 866 (1993); Moran, E. FASEB J. 7, 880 (1993); Vousden, K. FASEB J. 7, 872 (1993)).
The present inventors have shown that efficient replication of a plant geminivirus requires the integrity of an LXCXE amino acid motif in the viral RepA protein and that RepA can interact with members of the human Rb family in yeast (Xie, Q., Suxc3xa1rez-Lxc3x3pez, P. and Gutixc3xa9rrez, C. EMBO J. 14, 4073 (1995). The presence of the LXCXE motif in plant D-type cyclins has also been reported (Soni, R., Carmichael, J. P., Shah, Z. H. and Murray, J. A. H. Plant Cell 7, 85-103 (1995)).
The present inventors have now identified characteristic sequences of plant Rb proteins and corresponding encoding polynucleotides for the first time, isolated such a protein and polynucleotide, and particularly have identified sequences that distinguish it from known animal Rb protein sequences. The inventors have determined that a known DNA sequence from the maize encoding a vegetable Rb plant protein and is hereinafter called ZmRb1. ZmRb1 has been demonstrated by the inventors to interact in yeasts with RepA, a plant geminivirus protein containing LXCXE motif essential for its function. The inventors have further determined that geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the human Rb family.
Significantly the inventors work suggests that plant and animal cells may share fundamentally similar strategies for growth control, and thus human as well as plant Rb protein such as ZmRb1 will be expected to have utility in, inter alia, plant therapeutics, diagnostics, growth control or investigations and many such plant proteins will have similar utility in animals.
In a first aspect of the present invention there is provided the use of retinoblastoma protein in controlling the growth of plant cells and/or plant viruses. Particularly, the present invention provides control of viral infection and/or growth in plant cells wherein the virus requires the integrity of an LXCXE amino acid motif in one of its proteins, particularly, e. g., in the viral RepA protein, for normal reproduction. Particular plant viruses so controlled are Geminiviruses.
A preferred method of control using such proteins involves applying these to the plant cell, either directly or by introduction of DNA or RNA encoding for their expression into the plant cell which it is desired to treat. By over expressing the retinoblastoma protein, or expressing an Rb protein or peptide fragment thereof that interacts with the LXCXE motif of the virus but does not affect the normal functioning of the cell, it is possible to inhibit normal virus growth and thus also to produce infection spreading from that cell to its neighbors.
Alternatively, by means of introducing anti-sense DNA or RNA in plant cells in vectors form that contain the necessary promoters for the DNA or RNA transcription, it will be possible to exploit the well known anti-sense mechanism in order to inhibit the expression of the Rb protein, and thus the S-phase. Such plants will be of use, among other aspects to replicate DNA or RNA until high levels, e.g. in yeasts. The methods to introduce anti-sense DNA in cells are very well known for those skilled in the art: see for example xe2x80x9cPrinciples of gene manipulationxe2x80x94An introduction to Genetic Engineering (1994) R. W. Old and S. B. Primrose; Oxford-Blackwell Scientific Publications Fifth Edition p398.
In a second aspect of the present invention there is provided recombinant nucleic acid, particularly in the form of DNA or cRNA (mRNA), encoding for expression of Rb protein that is characteristic of plants. This nucleic acid is characterised by one or more characteristic regions that differ from known animal Rb protein nucleic acid and is exemplified herein by SEQ ID No 1, bases 31-2079.
The DNA or RNA can have a sequence that contains the degenerated substitution in the nucleotides of the codons in SEQ ID No. 1, and in where the RNA the T is U. The most preferred DNA or RNA are capable of hybridate with the polynucleotide of the SEQ ID No. 1 in conditions of low stringency, preferably being the hybridization produced in conditions of high stringency.
The expressions xe2x80x9cconditions of low stringencyxe2x80x9d and xe2x80x9cconditions of high stringencyxe2x80x9d are understood by those skilled, but are conveniently exemplified in U.S. Pat. No. 5,202,257, Col-9-Col 10. If some modifications were made to lead to the expression of a protein with different amino acids, preferably of the same kind of the corresponding amino acids to the SEQ ID No 1; that is, are conservative substitutions. Such substitutions are known by those skilled, for example, see U.S. Pat. No. 5,380,712, and it is only contemplated when the protein has activity with retinoblastoma protein.
Preferred DNA or CRNA encodes for a plant Rb protein having A and B pocket sub-domains having between 30% and 75% homology with human Rb protein, particularly as compared with p130, more preferably from 50% to 64% homology. Particularly the plant Rb protein so encoded has the C706 amino acid of human Rb conserved. Preferably the spacer sequence between the A and B pockets is not conserved with respect to animal Rb proteins, preferably being less than 50% homologous to the same region as found in such animal proteins. Most preferably the protein so encoded has 80% or more homology with that of SEQ NO 2 of the sequence listing attached hereto, still more preferably 90% or more and most preferably 95% or more. Particularly provided is recombinant DNA of SEQ ID No 1 bases 31 to 2079, or the entire SEQ ID No 1, or corresponding RNAS, encoding for maize CDNA clone encoding ZmRb1 of SQ ID No 2.
In a third aspect of the present invention there is provided the protein expressed by the recombinant DNA or RNA of the second aspect, novel proteins derived from such DNA or RNA, and protein derived from naturally occurring DNA or RNA by mutagenic means such as use of mutagenic PCR primers.
In a fourth aspect there are provided vectors, cells and plants and animals comprising the recombinant DNA or RNA of correct sense or anti-sense, of the invention.
In a particularly preferred use of the first aspect there is provided a method of controlling cell or viral growth comprising administering the DNA, RNA or protein of the second or third aspects to the cell. Such administration may be direct in the case of proteins or may involve indirect means, such as electroporation of plant seed cells with DNA or by transformation of cells with expression vectors capable of expressing or over expressing the proteins of the invention or fragments thereof that are capable of inhibiting cell or viral growth.
Alternatively, the method uses an expression vector capable of producing anti-sense RNA of the cDNA of the invention.
Another one of the specific characteristics of the plants protein and of the nucleic acids includes a N-terminal domain corresponding in sequence to the amino acids 1 to 90 of the SEQ ID No. 2 and a nucleotides sequence corresponding to bases 31 to 300 of the SEQ ID No. 1. These sequences are characterized by possessing less than 150 and less than 450 units that the animal sequences which possess more than 300 amino acids and 900 pairs of more bases.
The present invention will now be illustrated further by reference to the following non-limiting Examples. Further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these.